Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

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Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

International Journal of Molecular Sciences ◽

10.3390/ijms222313028 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13028

Author(s):

Richard Zimmermann ◽

Sven Lang ◽

Monika Lerner ◽

Friedrich Förster ◽

Duy Nguyen ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Import ◽

Protein Targeting ◽

Human Cells ◽

Protein Abundance ◽

General Role ◽

Differential Protein ◽

Er Targeting ◽

Dependent Pathway ◽

Er Membrane

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

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Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

10.20944/preprints202111.0414.v1 ◽

2021 ◽

Author(s):

Richard Zimmermann ◽

Sven Lang ◽

Monika Lerner ◽

Friedrich G Förster ◽

Duy Nguyen ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Import ◽

Protein Targeting ◽

Human Cells ◽

Protein Abundance ◽

General Role ◽

Differential Protein ◽

Er Targeting ◽

Dependent Pathway ◽

Er Membrane

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of about 10,000 different soluble and membrane proteins in humans. It involves co- or post-translational targeting of precursor polypeptides to the ER and their subsequent membrane insertion or translocation. So far, three pathways for ER targeting of precursor polypeptides plus four pathways for ER targeting of mRNAs were described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in targeting and, putatively, inserting monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose if this pathway may play a more general role in ER protein targeting, i.e. represents a fourth pathway for ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach, which involves label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells and differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3-clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices and belonging to the secretory pathway were also negatively affected by PEX3-deficiency, which may suggest compromised collagen biogenesis as a hitherto unknown contributor to organ failures in the respective Zellweger patients.

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Translational regulation of Pmt1 and Pmt2 by Bfr1 affects unfolded protein O-mannosylation

10.1101/847095 ◽

2019 ◽

Author(s):

Joan Castells-Ballester ◽

Natalie Rinis ◽

Ilgin Kotan ◽

Lihi Gal ◽

Daniela Bausewein ◽

...

Keyword(s):

Quality Control ◽

Translational Regulation ◽

Secretory Pathway ◽

Rna Binding ◽

Protein Quality ◽

Brefeldin A ◽

Protein Quality Control ◽

Ribosome Profiling ◽

Protein Abundance ◽

Unfolded Protein

ABSTRACTO-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding GFP as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance, but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

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Using a Label Free Quantitative Proteomics Approach to Identify Changes in Protein Abundance in Multidrug-Resistant Mycobacterium tuberculosis

Indian Journal of Microbiology ◽

10.1007/s12088-015-0511-2 ◽

2015 ◽

Vol 55 (2) ◽

pp. 219-230 ◽

Cited By ~ 10

Author(s):

Truong Quoc Phong ◽

Do Thi Thu Ha ◽

Uwe Volker ◽

Elke Hammer

Keyword(s):

Mycobacterium Tuberculosis ◽

Quantitative Proteomics ◽

Multidrug Resistant ◽

Protein Abundance ◽

Label Free ◽

Proteomics Approach

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EBprotV2: A Perseus Plugin for Differential Protein Abundance Analysis of Labeling-Based Quantitative Proteomics Data

Journal of Proteome Research ◽

10.1021/acs.jproteome.8b00483 ◽

2018 ◽

Vol 18 (2) ◽

pp. 748-752 ◽

Cited By ~ 1

Author(s):

Hiromi W. L. Koh ◽

Yunbin Zhang ◽

Christine Vogel ◽

Hyungwon Choi

Keyword(s):

Quantitative Proteomics ◽

Protein Abundance ◽

Proteomics Data ◽

Differential Protein

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Differential Protein Expression Profiles in Estrogen Receptor-Positive and -Negative Breast Cancer Tissues Using Label-Free Quantitative Proteomics

Genes & Cancer ◽

10.1177/1947601910365896 ◽

2010 ◽

Vol 1 (3) ◽

pp. 251-271 ◽

Cited By ~ 29

Author(s):

K. Rezaul ◽

J. K. Thumar ◽

D. H. Lundgren ◽

J. K. Eng ◽

K. P. Claffey ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Quantitative Proteomics ◽

Expression Profiles ◽

Label Free ◽

Differential Protein Expression ◽

Differential Protein ◽

Estrogen Receptor Positive ◽

Protein Expression Profiles ◽

Cancer Tissues

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DEqMS: A Method for Accurate Variance Estimation in Differential Protein Expression Analysis

Molecular & Cellular Proteomics ◽

10.1074/mcp.tir119.001646 ◽

2020 ◽

Vol 19 (6) ◽

pp. 1047-1057 ◽

Cited By ~ 7

Author(s):

Yafeng Zhu ◽

Lukas M. Orre ◽

Yan Zhou Tran ◽

Georgios Mermelekas ◽

Henrik J. Johansson ◽

...

Keyword(s):

Mass Spectrometry ◽

Statistical Analysis ◽

Protein Expression ◽

Statistical Methods ◽

Expression Analysis ◽

Quantitative Proteomics ◽

Data Sets ◽

Label Free ◽

Differential Protein Expression ◽

Differential Protein

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all data sets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several data sets including E. coli proteome spike-in data, using both label-free and TMT-labeled quantification. Compared with previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared with other statistical methods in both label-free and labeled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

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Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

International Journal of Plant Genomics ◽

10.1155/2012/494572 ◽

2012 ◽

Vol 2012 ◽

pp. 1-17 ◽

Cited By ~ 94

Author(s):

Cosette Abdallah ◽

Eliane Dumas-Gaudot ◽

Jenny Renaut ◽

Kjell Sergeant

Keyword(s):

Quantitative Proteomics ◽

Protein Extraction ◽

State Of The Art ◽

Rapid Expansion ◽

Protein Abundance ◽

Label Free ◽

Genome Sequences ◽

Two Dimensional Gel Electrophoresis ◽

Green Tissue ◽

Peptide Fractionation

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

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